[Objective] The aim was to establish the optimal rye-specific PCR reaction system for rye.[Method] The ordinary wheat ＂Chinese Spring＂,S165,rye,octoploid triticale and hexaploid triticale were used as materials to c...[Objective] The aim was to establish the optimal rye-specific PCR reaction system for rye.[Method] The ordinary wheat ＂Chinese Spring＂,S165,rye,octoploid triticale and hexaploid triticale were used as materials to carry out study on the effect of the amount of template DNA,primers,dNTPs,Mg2＋ concentrations,Taq DNA polymerase and annealing temperature on the rye-specific PCR reaction system of rye.[Result] The genomic DNA extracted by modified CTAB DNA extraction method showed high quality,which was satisfied for the PCR reaction template.The rye-specific PCR reaction system was 25 μl,including 10 × buffer solution,1.5 mmol/L MgCl2,200 μmol/L dNTP,40 ng primers,40-60 ng DNA template and 1 U Taq DNA polymerase.[Conclusion] The optimal rye-specific PCR reaction system was established,which provided basis for the identification of exogenous germplasm of rye in wheat background.展开更多
With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oler...With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oleracea were Optimized. The results showed that there were 8 primers suitable for ISSR-PCR of P. oleracea. The optimal reaction system had a volume of 25 μl, including 2 x Taq Platinum PCR Master Mix 12.5 μl, primer 2 μl, ddH20 9.5 μl, and DNA template 1μl. The optimized ISSR-PCR of P. oleracea was started with pre-denaturation at 94 ℃ for 360 s, followed by 30 cycles of denaturation at 94 ℃ for 60 s, annealing at 54 ℃ for 60 s and extension at 72 ℃ for 90 s, and completed by extension at 72 ℃ for 300 s.展开更多
以博白大果油茶Camellia gigantocarpa Hu et Huang为材料,通过单因素实验设计和正交设计L18(35)对影响PCR反应体系的主要成分及PCR扩增程序进行优化。建立了稳定的、可重复的博白大果油茶ISSR-PCR扩增反应体系。在20μL的反应体系中,M...以博白大果油茶Camellia gigantocarpa Hu et Huang为材料,通过单因素实验设计和正交设计L18(35)对影响PCR反应体系的主要成分及PCR扩增程序进行优化。建立了稳定的、可重复的博白大果油茶ISSR-PCR扩增反应体系。在20μL的反应体系中,Mg2+浓度为2.5mmol/L,模板DNA用量为20ng,Taq DNA聚合酶为1.75U,dNTPs浓度为0.25mmol/L,引物浓度为0.6mmol/L。反应程序为:94℃预变性4min,94℃变性30s,50℃退火45s,72℃延伸90s,35个循环,72℃延伸7min,4℃保存。展开更多
基金Supported by Young Academic Backbone Support Program of Heilongjiang Province(1152G047)Student Innovation Project of Mudanjiang Normal University~~
文摘[Objective] The aim was to establish the optimal rye-specific PCR reaction system for rye.[Method] The ordinary wheat ＂Chinese Spring＂,S165,rye,octoploid triticale and hexaploid triticale were used as materials to carry out study on the effect of the amount of template DNA,primers,dNTPs,Mg2＋ concentrations,Taq DNA polymerase and annealing temperature on the rye-specific PCR reaction system of rye.[Result] The genomic DNA extracted by modified CTAB DNA extraction method showed high quality,which was satisfied for the PCR reaction template.The rye-specific PCR reaction system was 25 μl,including 10 × buffer solution,1.5 mmol/L MgCl2,200 μmol/L dNTP,40 ng primers,40-60 ng DNA template and 1 U Taq DNA polymerase.[Conclusion] The optimal rye-specific PCR reaction system was established,which provided basis for the identification of exogenous germplasm of rye in wheat background.
文摘With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oleracea were Optimized. The results showed that there were 8 primers suitable for ISSR-PCR of P. oleracea. The optimal reaction system had a volume of 25 μl, including 2 x Taq Platinum PCR Master Mix 12.5 μl, primer 2 μl, ddH20 9.5 μl, and DNA template 1μl. The optimized ISSR-PCR of P. oleracea was started with pre-denaturation at 94 ℃ for 360 s, followed by 30 cycles of denaturation at 94 ℃ for 60 s, annealing at 54 ℃ for 60 s and extension at 72 ℃ for 90 s, and completed by extension at 72 ℃ for 300 s.
文摘目的建立鉴别鱼腥草Houttuynia cordata与百部还魂Gymnotheca chinensis的特异性PCR方法。方法采集不同产地的鱼腥草与百部还魂样品各8份,所有样品提取总DNA。通过对其mat K片段进行扩增、测序,进行同源比对后根据其变异位点设计特异鉴别引物,建立特异PCR鉴别方法,并通过加入SYBR Green I染料法对2种中药进行快速检测。另外,构建多重PCR体系,只经一个PCR反应,就能对鱼腥草与百部还魂进行快速分子鉴定。结果所构建特异PCR与多重PCR体系均能产生鱼腥草185 bp的特异鉴别条带,百部还魂389 bp的特异鉴别条带,SYBR Green I染料可进行快速检测方法。结论建立的鱼腥草与百部还魂2种中药的快速PCR鉴别方法简便、可靠。
文摘以博白大果油茶Camellia gigantocarpa Hu et Huang为材料,通过单因素实验设计和正交设计L18(35)对影响PCR反应体系的主要成分及PCR扩增程序进行优化。建立了稳定的、可重复的博白大果油茶ISSR-PCR扩增反应体系。在20μL的反应体系中,Mg2+浓度为2.5mmol/L,模板DNA用量为20ng,Taq DNA聚合酶为1.75U,dNTPs浓度为0.25mmol/L,引物浓度为0.6mmol/L。反应程序为:94℃预变性4min,94℃变性30s,50℃退火45s,72℃延伸90s,35个循环,72℃延伸7min,4℃保存。
